1Department of Biophysics and Biochemistry, Graduate School of Science, Hongo, Tokyo 113-0033, Japan.

Abstract

Schizosaccharomyces pombe ste11 encodes a high-mobility group family transcriptional activator that is pivotal in sexual development. Transcription of ste11 is induced by starvation of nutrients via a decrease of the cAMP-dependent protein kinase (PKA) activity. Here we report the identification of a novel transcription factor, Rst2p, that directly regulates ste11 expression. Cells in which the rst2 gene was disrupted expressed ste11 poorly and were sterile, and this sterility could be suppressed by artificial expression of ste11. Disruption of rst2 suppressed hypermating and hypersporulation in the PKA-null mutant, whereas overexpression of rst2 induced sexual development in the PKA-activated mutant. Cloning analysis indicated that Rst2p was a Cys(2)His(2) zinc-finger protein carrying 567 amino acid residues. Rst2p could bind specifically to a stress response element-like cis element located in the ste11 promoter region, which was important for ste11 expression. Meanwhile, transcription of ste11 was reduced significantly by a defective mutation in itself. An artificial supply of functional Ste11p circumvented this reduction. A complete Ste11p-binding motif (TR box) found in the promoter region was necessary for the full expression of ste11, suggesting that Ste11p is involved in the activation of ste11. We conclude that transcription of ste11 is under autoregulation in addition to control through the PKA-Rst2p pathway.

The structure of the rst2 gene. (A) A restriction map of the rst2 locus and functional analysis of the subclones. The arrow indicates the position and direction of the rst2 ORF. Open circles on the arrow represent the two Cys2His2 zinc-finger motifs. Arrowheads indicate the orientation of transcription from a cryptic promoter on the vector. Each subclone was examined for the ability to promote mating and sporulation in JZ858 (cgs1Δ). The construct used to disrupt the rst2 gene is shown at the bottom. Restriction sites are abbreviated as follows: Cl, ClaI; E, EcoRI; H, HindIII; Hc, HincII; Kp, KpnI; N, NdeI; Sac, SacI; Sal, SalI; and Sp, SphI. (B) The nucleotide sequence of a 3.8-kb SacI–SphI fragment carrying rst2 and the amino acid sequence of the deduced gene product. The two zinc-finger motifs are underlined. Amino acid residues that may be phosphorylated by PKA are italicized. (C) Comparison of zinc-finger motifs among Rst2p and its close homologues. The two zinc-finger motifs of Rst2p (residues 66–128) are aligned with S. cerevisiae Adr1p (residues 100–155; ), S. cerevisiae Mig1p (residues 34–90; ), S. cerevisiae Msn2p (residues 643–698), S. cerevisiae Msn4p (residues 569–624; ), and the human EGR1 gene product (residues 364–419; ). Amino acid residues identical to those of Rst2p are shown in white against black. Conserved amino acids are shown in white against gray. The number of fingers assigned for each individual protein is indicated in parentheses. Asterisks indicate cysteine and histidine residues conserved in the zinc-finger motif.

Necessity of rst2 for the transcription of ste11. Expression of ste11 was examined in the rst2+ (JY333) and rst2Δ (JX233) strains by Northern blot analysis. Total RNA was prepared from wild-type cells (lanes 1 and 2) and rst2Δ cells (lanes 3 and 4), either growing vegetatively (N+; lanes 1 and 3) or being starved for nitrogen (N−; lanes 2 and 4). rRNA stained with ethidium bromide is shown in the lower panel as loading controls. The relative intensity of ste11 transcription in each lane was calculated as described in MATERIALS AND METHODS and is presented under the top panel.

Suppression of the hypersporulation phenotype of the pka1Δ strain by disruption of rst2. The homothallic wild-type strain JY450 (A), the rst2Δ strain JX231 (B), the pka1Δ strain JZ633 (C), and the pka1Δ rst2Δ strain JX239 (D) were grown on malt extract agar medium for 2 d at 30°C, and cells were photographed under the phase-contrast microscope. Bar, 10 μm.

The ste11 promoter region. (A) Scheme and nucleotide sequence of the promoter region of the ste11 gene. The SphI–BamHI segment, shown by a bold line, represents the 1.4-kb region newly sequenced in this study. Two TR boxes therein are indicated by open ovals. The open square represents UASst, which encloses a STRE-like element core 1. The same six-base motif in the upstream is denoted as core 2. The major transcription start site, which is assigned as position +1 for the nucleotide numbering, is indicated by the asterisk. Two hatched ovals in the downstream represent incomplete TR boxes noted previously. UASst, core 2, TR1, and TR2 are underscored by straight lines in the nucleotide sequence, whereas the incomplete TR boxes are underscored by dashed lines. Restriction sites indicated are B, BamHI; EV, EcoRV; N, NdeI, and Sp, SphI. (B) Assignment of the major transcription start site for ste11. The 5′ ends of the ste11 transcripts were determined by primer extension analysis. Total RNA prepared from cells either starved for nitrogen (lane 1) or growing logarithmically (lane 2) was used as the template. A single major start site (closed arrowhead) and an adjacent minor start site (open arrowhead) were detected.

DNase I footprint analysis of the ste11 promoter region. (A) Left panel, The coding strand of the ste11 promoter region, labeled and processed as described in MATERIALS AND METHODS, was incubated with either 1.0 μg (lane 1) or 0.2 μg (lane 2) of recombinant Ste11p and either 0.3 μg (lane 7) or 1.5 μg (lane 8) of recombinant Rst2p. Each sample was subjected to DNase I digestion. Lanes 3 and 6 represent control analysis with no protein addition. Lanes 4 and 5 represent the G and the purine ladders, respectively. Right panel, Similar analysis was done with the noncoding strand. DNA was labeled and incubated with either no (lane 10) or 0.3 μg (lane 11) of recombinant Rst2p before DNase I digestion. Lane 9 represents the G ladder. Nucleotides protected from DNase I digestion are indicated by parentheses. (B) A summary of the protected sequences.

Specific binding of Rst2p to UASst in vitro. Recombinant Rst2p, either carrying the two zinc-finger motifs or lacking them, was incubated with labeled DNA probes, and formation of a protein–DNA complex was examined by gel mobility shift assay. (A) The protein with the zinc-finger motifs was mixed with a wild-type probe (WTa) in gradually increasing amounts (lane 3, 0.01 μg; lane 4, 0.1 μg; and lane 5, 1 μg of protein). Lane 6 is the same as lane 5 except for the addition of 10 mM 1,10-phenanthroline. Lane 7 is the same as lane 6 but it further accepted 0.5 mM zinc sulfate. Lanes 8–11 are the same as lane 5 except that cold competitor oligonucleotides were added to them in the molar ratios, as indicated in the panel. Lane 1 represents a mock experiment with no protein addition. Binding of the wild-type probe to the protein with no zinc-finger motifs (1 μg of protein) was examined in lane 2, and binding of a mutant probe (Ma) to the intact protein was examined in lane 12. (B) Mutational dissection of the binding sequence. A wild-type probe (WTb) was incubated with 0.01 μg of the protein carrying the zinc-finger motifs, together with (lanes 3 and 4) or without (lane 2) cold competitors. Mock reaction with no protein addition (lane 1) and binding of mutant probes (lane 5, Mb1; lane 6, Mb2; and lane 7, Mb3) to the protein were also examined.

Core 1 and TR1 are essential cis elements for the promoter function of ste11. Transcription of the ste11–mei2 fusion gene driven by variously modified ste11 promoters was examined by Northern blot analysis. Cells harboring each reporter construct on a plasmid were harvested either during vegetative growth (+nitrogen) or after being starved of nitrogen (−nitrogen). RNA was prepared from each sample and analyzed by the mei2 probe. (A) Lanes 1 and 2, expression from the wild-type promoter (pDM+); lanes 3 and 4, mutant in core 1 (pDM3); lanes 5 and 6, mutant in core 2 (pDM4); and lanes 7 and 8, mutant in both core 1 and core 2 (pDM34). (B) Lanes 9 and 10, expression from the wild-type promoter (pDM+); lanes 11 and 12, mutant in TR1 (pDM1); lanes 13 and 14, mutant in TR2 (pDM2); and lanes 15 and 16, mutant in both TR1 and TR2 (pDM12). The major transcript of the reporter gene is indicated by the arrowhead. rRNA stained with ethidium bromide is shown in the lower panels as loading controls. The relative intensity of transcription is presented under the top panels.

Activation of ste11 transcription by Ste11p. The multicopy vector pDB248′ (lanes 1, 2, 5, 6, 9, and 10) and the ste11-expressing plasmid pDB-ste11+ (lanes 3, 4, 7, 8, 11, and 12) were introduced into the wild-type strain JY450 (lanes 1–4), the ste11 point mutant JY858 (lanes 5–8), or the ste11Δ strain JZ396 (lanes 9–12). Total RNA was prepared from each strain either growing logarithmically (odd-numbered lanes) or starved for nitrogen (even-numbered lanes) and analyzed by Northern blotting. Transcripts from the chromosomal ste11 allele (wild type or ste11-029) and truncated transcripts from the episomal allele on pDB-ste11+ are indicated by arrowheads. rRNA stained with ethidium bromide is shown in the lower panel as loading controls. The relative intensity of transcription is presented under the top panels.